Olfactory Receptors
The genes coding for olfactory receptors (ORs) represent the largest family of genes (3% of the whole genome) in the human body dedicated to a single physiological function. These ORs belong to the superfamily of G protein coupled receptors (GPCRs). GPCRs are membrane receptors usually located at the surface of many different cell types. The common features of these receptors consist of seven transmembrane spans that form a barrel within the cell membrane and in their capacity to interact with heterotrimeric GTPase and thereby transducing a signal upon binding of their activators.
In the human genome, about 900 sequences containing characteristic signatures of olfactory receptors have been found. However, 60% of these appear to encode non-functional pseudogenes, thereby leaving humans with about 380 different OR proteins. ORs are characterized by 6 conserved amino acid motifs in their sequence. The first is the FILLG motif (SEQ ID No. 17) located in the extracellular N-terminal end of the receptor. It corresponds to a highly conserved phenylalanine and glycine separated by 3 variable but mostly hydrophobic amino acids. The other motifs include LHTPMY (SEQ ID No. 18) in intracellular loop 1, MAYDRYVAIC (SEQ ID No. 19) at the end of transmembrane domain 3 and the beginning of intracellular loop 2, SY (SEQ ID No. 20) at the end of transmembrane domain 5, FSTCSSH (SEQ ID No. 21) in the beginning of transmembrane domain 6, and PMLNPF (SEQ ID No. 22) in transmembrane domain 7.
The mammalian ORs are usually subdivided in two distinct classes. Class 1 ORs, also called fish-like receptors, form a homogenous group that is more closely related to ORs found in fish and are therefore assumed to represent a conserved relic maintained throughout the evolution of the vertebrates. The persistence of this group of ancestral ORs suggests that they play an important role in mammalian chemical perception. In humans, class 1 ORs encompass 68 non-pseudogenic sequences that correspond to potential functional proteins. These receptors share several characteristic domains in their sequence that allows their classification as “class 1” ORs. It is also to be noted that some amino acids located in the transmembrane domains are highly conserved within the members of the fish-like ORs. In contrast to the fish-like ORs, class 2 ORs first appeared in tetrapode vertebrates and expanded to form the majority of the OR repertoire presently known in humans. Class 2 ORs probably represent an adaptation to the terrestrial life where the detection of airborne odorants is required.
Mechanisms of Odor Perception
Each OR is able to interact with different molecules, and each odorant molecule can activate more than one OR. Thus, odor perception does not rely on the simple activation of a single OR, but rather on multiple activations of several ORs. An odor (which can be a single molecule or a mixture) is paired with a unique set of activated ORs that are sufficient for its discrimination and characterization. Odorant concentration can dramatically affect the profile of an odor as some additional ORs may be recruited (high concentration) or not activated (low concentration). Therefore, the set of activated ORs will differ for different odor concentrations, leading to varying odor perceptions. With a pool of 380 ORs, the number of possible combinations is almost infinite, thus explaining the outstanding discrimination properties of the olfactory system. Odorant receptors are expressed in specialized olfactory sensory neurons (OSNs) located at the top of the nasal cavity in a small area that constitutes the olfactory epithelium. Filiform extensions at one end of these cells contain the ORs on their surface and float in the nasal mucus where the odorants are dissolved. At the opposite end, the OSN extends its axon across the ethmoid bone at the base of the cranium to connect to the olfactory bulb a small region of the brain dedicated to the integration of the olfactory stimuli. An outstanding feature of the tens of millions of OSNs scattered throughout the olfactory epithelium is that each one expresses only one of the about 400 OR genes available in the human genome. The OSNs expressing the matching gene connect their axons to the same subregion of the olfactory bulb forming a structure called a glomerulus. It is from this organization of OSNs that the coding of an odor by a specific set of activated ORs is translated geographically in the bulb by a corresponding pattern of activated glomeruli. This information is further transmitted to the olfactory area of the cortex where it is decoded and analyzed. In OSNs, triggering of the OR promotes the activation of an olfactory-specific G protein (Galpha-olf) that stimulates a type III adenylate cyclase to produce cyclic AMP; this plays the role of a second messenger. Upon binding to a cAMP-gated cation channel, this messenger induces the entry of calcium into the cell. Calcium causes the opening of another channel that promotes the exit of chloride ions, and hence triggers an action potential of the neuron leading to a signal to the respective brain area.
Characterisation of Odorant Molecules with ORs
Cultured cell lines have been widely used to characterize and study receptors of interest in both academic and industrial contexts. This approach involves introduction of the corresponding gene into the cells, and subsequent promotion of its stable or transient overexpression. The activity of the receptor can be monitored using a functional assay. The use of easy-to-culture cell lines along with easy-to perform functional assays facilitates several thousand measurements per day. Typically, in the pharmaceutical industry, it is common to test libraries of 1,000,000 compounds per day on non-olfactory receptors. In the aftermath of OR discovery, several attempts were made to express ORs in the cell lines suitable for the expression of non-olfactory receptors, but they remained largely unsuccessful. The reason for such a setback can be found, not in the failure of the cell to produce the receptor, but rather in its inability to send the receptor to the surface of the cell. A technique aimed at improving the functional expression of ORs requires engineering a conventional cell line to make it suitable for OR expression. In fact, it had long been suspected that correct expression and targeting of the OR at the cell surface requires an OSN-specific intracellular machinery that is absent in a non-olfactory cell line. Thorough analysis of the expression in OSNs revealed two members of a new family of proteins that are specific to this sensory cell. When co-introduced into a conventional cell line along with a model OR, the so-called receptor transport proteins 1 and 2 (RTP1 and RTP2) enhanced both the cell surface expression and the response of the receptor to its cognate odorants. The production of cAMP arising in the cell upon activation of the OR by its odorant molecules may be detected by an indirect approach that consists of the use of a reporter gene, as described in (Saito et al., 2004 Cell Vol. 119, 679-691). This gene is placed under the control of a cAMP inducible promoter and is expressed only upon induction by cAMP. Different genes can be used for this purpose, but one of the most popular ones encodes the light-producing protein luciferase. While cleaving its substrate, luciferin, this enzyme releases light that is readily detected and quantified. The intensity of light emitted reflects the amount of luciferase produced, which is proportional to the cAMP increase and therefore directly related to the activity of the receptor. One of the advantages of reporter gene assays is dependent upon the signal amplification between receptor activation and reporter production. This makes the assay particularly sensitive to weak responses that can hardly be detected by other functional assays.
Other functional assays have also been used to demonstrate the activation of an OR by its odorant ligand. One of these assays consists in monitoring the increase in cytosolic calcium that occurs upon activation of the receptor intracellular calcium increase (Krautwurtz D. et al. 1998. Cell 95, 917-26).
So far, the identification of odorant activators has only been reported for few mouse odorant ORs. Example of mouse OR deorphanization are given in Malnic et al., 1999, Cell 96, 713-23; Saito H. et al. 2009. Sci. Signal. 2, 1-14).
The identification of human OR activators has also been reported. Examples of deorphanized human ORs are e.g. given in Fujita Y et al. 2007. J. Recept. Signal. Transduct. Res. 27, 323-34; Keller A. et al. 2007. Nature 449, 468-72; Matarazzo V. et al. 2005. Chem. Senses 30, 195-207; Saito H. et al. 2009. Sci. Signal. 2, 1-14; Sanz G. et al. 2005. Chem. Senses 30, 69-80; Schmiedeberg K. et al. 2007. J Struct. Biol. 159, 400-12.; Shirokova E. et al. 2004. J. Biol. Chem. 280, 11807-15.; Spehr M. et al. 2003. Science 299, 2054-58.; Wetzel, K. et al. 1999. J. Neurosci. 19, 7426-33; Sallmann et al. PCT No. WO2006/094704.
For several of the receptors, more than one ligand has been identified. Odorants activating the same OR can belong to different odorant families such as alcohol, aldehyde, esters, etc (Sanz G. et al. 2005. Chem. Senses 30, 69-80; Saito H. et al. 2009. Sci. Signal. 2, 1-14).
Body Malodors
In our modern society, odors released by the human body, and more precisely in the sweat, are often considered as unpleasant or even offensive. Significant efforts have been made by the cosmetic industry to counteract the perception of these odors. Amongst the various categories of molecule present in human sweat, short chain carboxylic acids are of particular importance. Indeed, more than 50 different acids have been identified in sweat. A series of acids is assumed to participate in or to be important for the genesis of malodour (Table 1). For example, 3-hydroxy-3-methylhexanoic acid or (E)-3-methyl-2-hexenoic possess a pungent odor and both are known to be important contributors to axillary malodor (Zeng et al. 1991; J. Chem. Ecolog. Vol. 17 pp 1469-1492; Gautschi et al., 2007, Chimia, Vol. 61 pp 27-32). Isovaleric acid, another short chain carboxylic acid, has been identified as the major molecule responsible for the characteristic cheesy odor released by sweating feet (Ara et al. 2006. Can. J. Microbiol. Vol. 52 pp 357-364). Acids are not directly produced by the apocrine glands. They appear under the form of glutamine conjugates and are released under the action of skin bacteria enzymes. The abundance of several malodorants may therefore vary from one individual to another, depending on the composition of his bacterial flora.
TABLE 1Carboxylic acids contributing to sweat malodorMoleculeReferencesOdor descriptoracetic acid1, 2, 3sharp pungent sour vinegarpropanoic acid2, 3pungent acidic cheesy vinegarbutanoic acid1, 2, 3sharp acetic cheese butter fruitIsovaleric acid2, 3sour stinky feet sweaty cheese tropicalpentanoic acid3sickening putrid acidic sweaty rancidhexanoic acid2, 3, 4, 5sour fatty sweat cheese2-methylhexanoic acid4acid, animalic, honey, civet, sweet3-methylhexanoic acid4sweaty, butyric(E)-3-methyl-2-4, 6acid, sweaty, fruity, fatty, hexenoic acidlabdanum, hay, soupy3-hydroxy-3-6pungent sweatymethylhexanoic acidHeptanoic acid4, 5rancid sour cheesy sweat2-methylheptanoic acid4sour-fruity, sweet, slightly fatty-oilyOctanoic acid2, 3, 4, 5fatty waxy rancid oily vegetable cheesy4-ethyloctanoic acid4, 6costus, fatty, greasynonanoic acid4waxy dirty cheese cultured dairydecanoic acid3, 4acid, hot iron, metallic, waxy, soapy, metal, candleUndecanoic acid5waxy creamy cheese fatty coconutbenzoic acid2sweet; benzoin; powderyphenylacetic acid6sweet, animal-honey1. Yamazaki et al. (2010) Anti-Aging Medicine. 7(6): 60-652.2. Gallagher et al. (2008) Br. J. Dermatol. 159(4): 780-7913.3. Ara et al. (2006) Can. J. Microbiol. 52: 357-3644.4. Zeng et al. (1991) J. Chem. Ecol. 17(7): 1469-14925.5. Labows et al. (1999) Antiperspirants and Deodorants, 2nd Edition ed K. Laden, Cosmetic Science and Technology Series Vol. 20 Marcel Dekker Inc, New York, 59-826.6. Natsch et al (2006) Chem. & Biodiv. 3: 1-20
Different strategies have been developed to counteract sweat malodors. The most conventional ones consist in overpowering the malodor with a pleasant fragrance. In a more sophisticated approach, the fragrance is designed to harmonize well with the malodor and to shift the perception to a more pleasant character. In this case, the fragrance does not need to be a strong odorant by itself.
An alternative way for reducing malodor consists of limiting the production of odorant molecules. This can be achieved either by limiting the skin bacteria population with bacteriostatic agents or by blocking the enzymes responsible for the malodorant release. The development of antagonists and/or blockers that would specifically block the receptors for a malodor molecule can also be considered. An ideal blocker would have no odor per se, would not affect the bouquet and therefore would give a full creative freedom to perfumers.
In the present invention it has surprisingly been discovered that seven olfactory receptors belonging to class 1 of ORs are activated by carboxylic acids present in human sweat. This unexpected discovery allows the identification of compounds, which is of interest for the perfumer and flavorist companies. Indeed, the identified natural ligands of these seven olfactory receptors are known to be important constituents of sweat malodor. The identification and the use of blockers or antagonists of these olfactory receptors in a fragrance composition in order to modify the perception of sweat malodor represents an original concept that can open a new possibility for deodorant development.